Differentiation of Human Embryonic and Induced Pluripotent Stem Cells

ABSTRACT

The invention relates to culture systems, methods, and conditions that allow pluripotent undifferentiated hESCs or iPSCs to progressively and uniformly differentiate into cells of the chondrogenic lineage.

RELATED APPLICATION INFORMATION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/264,170, filed on Nov. 24, 2009.

FIELD OF THE INVENTION

The invention generally relates to methods for directing thedifferentiation of human embryonic and induced pluripotent stem cellsinto the cartilage lineage.

BACKGROUND OF THE INVENTION

Degenerative diseases of cartilage, e.g., osteoarthritis, are among themost prevalent and debilitating chronic health problems in the UnitedStates. Treatment of degenerative cartilage diseases is a particularclinical challenge because of the limited capacity of the tissue forself-repair. Because of their ability to differentiate into multiplecell types and their unlimited capacity for self-renewal, humanembryonic stem cells (hESCs) are a potentially powerful tool for therepair of cartilage defects. Fulfilling the potential of hESCs forrepair of diseased and damaged cartilage requires developing methods fordirecting their differentiation into the chondrogenic lineage.

Although several culture systems have been developed in whichESC-derived cells differentiate to various degrees into chondrocytes, inthese systems chondrogenic differentiation is not uniform, andchondrocytes represent only a subpopulation of the cells thatdifferentiate, complicating utilization of the cell population forcartilage repair. Prior to the invention described below, most of thechondrogenic differentiation protocols utilized cells of embryoid bodies(EBs) derived from ESCs. A drawback of such earlier methods is that thecellular heterogeneity of EB-derived cells hinders the ability to obtainhomogeneous populations of chondrogenic cells that can be used forcartilage repair.

SUMMARY OF THE INVENTION

The invention solves many of the problem associated with such earliermethods and is based, in part, on culture systems and conditions thatpromote the rapid, direct, progressive, and substantially uniformdifferentiation of stem cells such as undifferentiated pluripotent humanembryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs oriPS cells) into the chondrogenic lineage without prior EB formation.Stem cells obtained from cord blood or amniotic fluid are also used inthe methods. The substantially pure chondrogenic cells at defined stagesof differentiation that are produced using the methods provide asolution to the problems and drawbacks of cell heterogeneity (withrespect to tissue type as well as stage of differentiation) associatedwith prior methods. The cell populations described herein aresubstantially free of non-chondrogenic cells, e.g., the populationscomprise at least 85%, 95-98%, and up to 99 or 100% chondrogenic cells.The invention also provides high density culture conditions andadditional procedures which promote the progression of thedifferentiation of ESCs into the chondrogenic lineage and whichcontribute to the uniformity of chondrogenic differentiation achieved.

Embryoid bodies are aggregates of cells derived from ESCs or from iPSCs.These aggregates or EBs are apparent upon visual inspection of thecultures. Upon aggregation (formation of EBs), differentiation isinitiated. EBs differentiate into multiple cell types derived from all 3germ layers of the embryo that give rise to all of the cell typespresent in an adult organism. Prior to the invention, a drawback ofearlier methods of producing populations of chondrogenic precursor cellsfrom hESC was contamination with non-chondrogenic cells, thosenon-chondrogenic cells having been generated from EB differentiationinto lineages other than the desired target lineage (chondrogeniclineage). Since the methods described herein avoid EB formation, theresulting chondrogenic precursor cells are substantially free fromcontaminating cells of other lineages. The methods solve the problem ofEBs in the culture differentiating into non-chondrogenic tissue/cells.Since earlier methods required removal of contaminating undesirablecells, the process described herein is more efficient, faster, and morecost-effective than previous methods.

A method for direct differentiation of human embryonic stem cells(hESCs) into chondrogenic cells is carried out by providing a populationof hESCs on a substrate, said population being substantially free ofEBs; detaching the hESCs from said substrate and dissociating saidhESCs; establishing a high-density micromass culture of the hESCs; andcontacting the population with a bone morphogenetic protein; wherein thehESCs directly differentiate into a substantially uniform population ofchondrogenic cells. Similarly, a method for direct differentiation ofinduced pluripotent stem cells (iPSCs) into chondrogenic cells iscarried out by providing a population of iPSCs on a substrate, saidpopulation being substantially free of EBs; detaching the iPSCs from thesubstrate and dissociating the iPSCs; establishing a high-densityculture of the iPSCs; and contacting the iPSCs with a bone morphogeneticprotein thereby directing the differentiation of iPSCs into thechondrogenic lineage. The iPSCs directly differentiate into asubstantially uniform population of chondrogenic cells.

In one aspect, the culture systems and conditions described hereinpromote substantially uniform cartilage differentiation within less thanabout 1 or 2 weeks, e.g., about 2 days to about 3 days; about 3 days toabout 4 days; about 5 days to about 6 days; about 6 days to about 7days; about 7 days to about 8 days; about 8 days to about 9 days; about9 days to about 10 days; about 10 days to about 11 days; about 11 daysto about 12 days; about 12 days to about 13 days, or about 13 days toabout 14 days. In another aspect, the culture systems and conditions ofthe invention promote quite uniform differentiation such that at leastabout 50-100% of undifferentiated pluripotent human embryonic stem cellsdifferentiate into the chondrogenic lineage, e.g., at least about50-60%; at least about 60-70%; at least about 70-80%; at least about80-90%; or at least about 90-100% of undifferentiated pluripotent humanembryonic stem cells differentiate into the chondrogenic lineage.Preferably, the population comprises 95% chondrogenic cells; morepreferably, the population comprises 98% chondrogenic cells.

Stage of differentiation is described temporally or by expression of apanel of genetic markers. Timing (days) reflects the amount of timeafter the establishment of a high density culture of hESCs or iPSCs.Time zero is the point at which the cell concentration or density isadjusted to at least 1×10⁵ cells per 10 “2 day” cells are cells thathave been cultured for 2 days after the establishment of a high densitycolony. A high density culture comprises cells at a concentration of atleast 1×10⁵ per 10 μl. For example, 2-3 day cells are characterized asjust entering the cartilage lineage (i.e., chondroprogenitors), day 4cells are in an early phase of chondrogenic differentiation (i.e., earlychondrocytes), and 7-14 day cells have uniformly undergone overtdifferentiation into chondrocytes but have not undergone hypertrophicmaturation (i.e., the cells are characterized as fully differentiatedchondrocytes) (FIG. 14).

The invention describes the characterization of hESC-derived progenitorcells in different phases of the chondrogenic lineage, ranging fromcells just entering into the chondrogenic lineage to overtlydifferentiated chondrocytes. hESC-derived progenitor cells in differentphases of chondrogenic lineage are analyzed for their ability to repaircartilage using cell based tissue engineering therapies.

The conditions that promote the chondrogenic differentiation of hESCsalso promote the uniform cartilage differentiation of inducedpluripotent stem cells (iPSCs). The invention also provides methods ofdirect chondrogenic differentiation of iPSCs derived from patients. Forexample, iPSC-derived chondrogenic precursor cells (autologous cells)are used for patient-specific therapeutic approaches. In anotherexample, the methods are used to produce cells and cell lines to be usedas a research tool to elucidate underlying mechanisms of geneticdisorders of cartilage, such as chondrodysplasias, in order to studymechanisms of disease as well as to using the cells to screen fortherapeutic agents.

As discussed above, conditions that promote the chondrogenicdifferentiation of hESCs also promote the uniform cartilagedifferentiation of induced pluripotent stem cells (iPSCs), i.e., somaticcells that have been reprogrammed to a pluripotent state. Reliabledirect and uniform differentiation of iPSCs into the chondrogeniclineage permits patient-specific autologous cell therapy. Thus,iPSCs-derived chondrogenic progenitor cells, as well as hESC-derivedprecursors, are used to repair cartilage defects.

The culture systems and conditions of the invention avoid the cellularheterogeneity that complicates the use of cells derived from embryoidbodies (EBs) for cell-based cartilage repair therapies. Since themethods promote direct and uniform differentiation and circumvent the EBstage, they reliably yield cell populations at a defined stage ofdifferentiation, i.e., early stage versus late stage. Gene expressionprofiling enables the identification of hESC-derived progenitor cells indifferent stages of the chondrogenic lineage, ranging from cells justentering into the chondrogenic lineage to overtly differentiatedchondrocytes. In addition to continuing to differentiate, cells inearlier phases of the chondrogenic lineage (e.g., bearing earlycartilage markers such as Sox9) respond to local microenvironmentalsignals in situ after they are transplanted into a recipient and arethus desirable for participation in cartilage repair compared to cellsat late stages of the lineage. Cells in late stages of the chondrogeniclineage (e.g., bearing markers indicative of more overtly differentiatedcells cartilage markers such as aggrecan) are suitable for cartilagerepair/restoration procedures. Thus, unlike earlier methods that yieldeda heterogeneous population of cells at various stages of differentiationand different lineages, the present methods provide a source of stagedcells and permit fine tailoring of therapies based on the stage ofdifferentiation best suited to the pathological condition to be treated.

The invention also provides conditions and additional procedures whichenhance the progression of the differentiation of hESCs and iPSCs intothe chondrogenic lineage and which contribute to the uniformity ofchondrogenic differentiation achieved. These modifications include: (1)maintenance of the dissociated hESC in high density culture (absence ofEBs); (2) supplementation of the cultures with growth factors especiallyBMP-2 and TGFβ-1; (3) culturing hESCs and iPSCs on or within polymericor gelatinous substrates or scaffolds rather than on a feeder layer ofmouse embryonic fibroblasts; (4) using Accutase, TrypLE Select or otherenzymes rather than trypsin for dissociating the hESCs prior topreparing micromass cultures; and, (5) application of a Rho-associatedkinase (ROCK) inhibitor, which diminishes dissociation-inducedapoptosis, during the establishment of micromass cultures. Suitablepolymeric or gelatinous substrate or scaffold materials include basementmembrane substrates (e.g. Matrigel, Chondrogide); collagens or gelatins;hydrogels or sponges containing hyaluronan (e.g. Hyaffl 1 or HyStemC) orchitosan; or PEG (poly ethylene glycol) or PLGA (polylacticacidglyolyticacid) scaffolds; or any substrate containing extracellular matrixcomponents.

Specifically, the invention provides methods for directing thedifferentiation of human embryonic stem cells (hESCs) into thechondrogenic lineage by culturing hESCs in serum-free medium on asubstrate; detaching the hESCs from the substrate and dissociating thehESCs; culturing the hESCs as a high-density culture (micromass orpellet) in serum-free medium; and administering bone morphogeneticprotein-2 (BMP2; NG_(—)023233 (GI:300068920)), incorporated herein byreference) to the micromass culture medium. The hESCs are substantiallyfree of EBs; preferably the hESCs are completely free of EBs. The BMP2is administered 12, 24, 48, 72, or 96 hours after initiation of the hESChigh-density micromass culture. Preferably, the growth factor(s) areadded 48 hours after the establishment of high density cell cultures.Alternatively, BMP-2 and transforming growth factor-beta (TGFβ;NM_(—)000660 (GI:260655621)), incorporated herein by reference) areadded together. The concentration of BMP2 is in the range of 25-200ng/ml, and the concentration of TEGβ1 is in the range of 2.5-20 ng/ml.The ratio of BMP to TGF is approximately 5:1 to 20:1, with a preferredratio of about 10:1.

The hESCs are contacted with a dissociating agent such as trypsin,TrypLE Select, or Accutase prior to culturing the hESCs as ahigh-density micromass culture. Optionally, the substrate for hESCculture is a feeder layer comprising mouse embryonic fibroblasts (MEF).Alternatively, the substrate for hESC culture is a gelatinouscomposition such as artificial basement membrane material (e.g.,Matrigel) or other suitable substrate or scaffold. In one aspect, themethod further comprises administering transforming growth factor beta-1(TGFβ1) to the micromass culture medium. Optionally, the method furthercomprises the administration of a Rho-associated kinase (ROCK)inhibitor.

In one aspect, the hESCs differentiate into cells of the chondrogeniclineage within about 14 days, e.g., within about 10 days; within about 7days; within about 4 days or within about 3 days. The methods describedherein direct the differentiation of cells such that at least about 85%of the hESCs differentiate into cells of the chondrogenic lineage; e.g.,at least about 90%; at least about 95%; at least about 98%; at leastabout 99% or about 100%. The differentiation is preferably carried outin the absence of bovine articular chondrocytes, e.g., differentiatedbovine articular chondrocytes. The process is also carried out in theabsence of conditioned media from cells, or a cell line such as ahepatocarcinoma cell line.

The invention also provides methods for directing the differentiation ofinduced pluripotent stem cells (iPSCs) into the chondrogenic lineage byculturing iPSCs in serum-free medium on a substrate; detaching the iPSCsfrom the substrate and dissociating the iPSCs; culturing the iPSCs as ahigh-density micromass culture in serum-free medium; and administeringbone morphogenetic protein-2 (BMP2) to the micromass culture. The iPSCsare substantially free of EBs; preferably the iPSCs are completely freeof EBs. In one aspect, the iPSCs are derived from a patient with acartilage disorder. In one aspect, the cartilage disorder is achondrodysplasia.

The methods described above yield chondrogenic cells, which arecharacterized by a defined stage of differentiation and by being free ofEB-derived cells, e.g., those of non-chondrogenic lineage. For example,the population of chondrogenic cells is substantially free ofnon-chondrogenic cells, the population having been differentiated fromESCs or iPSCs as described above. In another example, the cellpopulation is synchronized or staged. A uniformly differentiatedpopulation of chondrogenic cells contains at least 85% (or 98%, 99%, or100%) of cells, which are characterized as being at a single definedstage of differentiation. For some clinical applications, the stage ofdifferentiation is relatively early, e.g., the population issubstantially free of fully differentiated chondrocytes. For example,the stage is selected from the group consisting of day 3 chondrogeniccells or day 4 chondrogenic cells. Utilization of cells in early phasesof the lineage are more responsive to local environmental signals thatpromote articular cartilage repair after implantation. Thus, a method ofrepairing or restoring cartilage is carried out by contacting damaged ordiseased cartilage with any of the cell populations described above.Similarly, a method of preventing or treating arthritis is carried outby administering to an articulating joint or joint space of anindividual (human or other animal, e.g., dog, cat, horse) any of thedescribed chondrogenic cell populations.

The invention includes methods of treating cartilage disorders ordefects such as those arising from injury or degeneration. Disorders tobe treated include traumatic injuries to articulating joints (e.g.,knees including anterior cruciate ligament (ACL) or other ligament tearsor ruptures, meniscus tears or fractures), elbows, shoulders (e.g.,rotator cuff injury), jaw (temporomandibular joint or TMJ disease) orfingers, and other cartilage or skeletal injuries as well as chronicconditions such as arthritis that develop with age includingosteoarthritis (OA), or with inflammation including rheumatoid arthritis(RA), or as a consequence of a traumatic injury, and affecting any ofthe articulating joints. The cells are useful for prevention ofarticular cartilage damage due to injury or chronic disease. The cellsare also useful for repair of fibrocartilage, ligment or meniscus injuryor degeneration. A method of therapy includes the steps of administeringthe cells produced by the methods herein to a patient with a cartilagedisorder, e.g., cartilage injury or arthritis such as osteoarthritis.Therapy also includes introduction of the cells produced by this methodto delay or prevent the onset of chronic articular cartilagedegeneration due to injury or disease. Methods of repairing cartilagedefects with chondrogenic cells produced by the methods involvedadministering the cells to the articulating joint or other targetlocation by implantation, injection, or infusion. For example, the cellsare administered arthroscopically. In some cases, the cells areadministered or implanted before, after or during a surgical orarthroscopic procedure to repair an associated defect or condition,e.g., cells are administered to a joint space in conjunction with an ACLrepair procedure, meniscus repair, rotator cuff repair, or otherprocedure. The cells are also useful in methods of regenerating fingersor limbs lost to traumatic injury due to accident or military conflict,or congenital defect.

A significant advantage of the methods is that they permit isolation ofsubstantially pure populations of cells at defined stages ofdifferentiation, e.g., at a stage where cells are just beginning todifferentiate (e.g., day 2 cells, day 3 cells, or day 4 cells) or fullydifferentiated chondrocytes (e.g., day 7-14 cells). Fully differentiatedchondrocytes are useful for numerous cartilage repair and restorationtherapies. Cells at earlier stages of differentiation are used for thesame purpose or for other purposes with the added advantage that theycolonize and undergo further differentiation as well as respond to localenvironmental signals in situ.

The cells produced using the methods described above are also useful todetermine the genetic basis of disease and to screen for therapeuticagents to be used to treat or reduce the severity of cartilagedisorders. For example, a method of identifying a gene involved in thedevelopment of a cartilage disorder is carried out by providing asubstantially uniform population of cells at a known stage ofdifferentiation (e.g., day 2-3 cells, day 4 cells, of fullydifferentiated cells) and detecting an increase or decrease in geneexpression compared to normal control cells. An increase or decrease ingene expression indicates that the differentially-expressed gene isinvolved in the development of the cartilage disorder. For example, thecells are iPSCs obtained from a patient who has been diagnosed withchondrodysplasia or achondroplasia.

An exemplary screening method to identify a therapeutic agent (e.g., acartilage promoting agent) to treat or reduce the severity of acartilage disorder is carried out by providing a substantially uniformpopulation of cells at a known stage of differentiation (e.g., day 2-3cells, day 4 cells, of fully differentiated cells) and contacting thepopulation with a candidate compound and detecting chondrogenesis. Anincrease in chondrogenesis in the presence of the said compound comparedto in the absence of the compound indicates that the compound promotesthe formation of cartilage. A decrease in chondrogenesis indicates thatthe compound inhibits the formation of cartilage.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publishedforeign patents and patent applications cited herein are incorporatedherein by reference. Genbank and NCBI submissions indicated by accessionnumber cited herein are incorporated herein by reference. All otherpublished references, documents, manuscripts and scientific literaturecited herein are incorporated herein by reference. In the case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photomicrograph showing a colony of undifferentiatedpluripotent H9 human embryonic stem cells (hESCs) cultured on a feederlayer of irradiated mouse embryonic fibroblasts.

FIG. 1B is a photomicrograph showing embryoid bodies (EBs) derived fromhESC colonies one day after their formation.

FIGS. 2A-D are photomicrographs showing Alcian blue stained day 21micromass cultures established from the cells of EBs: (A) untreatedcontrol culture; (B) bone morphogenetic protein-2 (BMP2)-treatedculture; (C) transforming growth factor_beta-1 (TGFβ1)-treated culture;and (D) culture treated with both BMP2 and TGFβ1.

FIGS. 3A-D are photomicrographs showing a time course of Alcian bluematrix accumulation in EB cell micromass cultures treated with both BMP2and TGFβ1.

FIGS. 4A-C are photomicrographs showing the effect of BMP2 onaccumulation of Alcian blue cartilage matrix and type II collagen inmicromass cultures established directly from undifferentiatedpluripotent hESCs: (A) Alcian blue stained day 14 untreated controlculture; (B) Alcian blue stained day 14 BMP2-treated culture; and (C) asagittal section through a day 14 BMP2-treated culture immunostainedwith a type II collagen antibody.

FIGS. 5A-F are photomicrographs showing a time course of Alcian bluematrix accumulation in BMP2-treated micromass cultures established fromundifferentiated pluripotent hESCs: (A-C) cultures were supplementedwith BMP2 in “chondrogenic medium” on day 2; (D-F) cultures wereprovided with “chondrogenic medium” lacking BMP2 on day 2, and thensupplemented with BMP2 on day 3.

FIGS. 6 a-D are photomicrographs showing Alcian blue stained day 14micromass cultures established from undifferentiated pluripotent hESCs:(A) untreated control culture; (B) BMP2-treated culture; (C)TGFβ1-treated culture; (D) culture treated with both BMP2 and TGFβ1.

FIGS. 7A-E are bar graphs showing Sox9, aggrecan, Col2a1, Col10a1, andosteopontin (OPN) (A) and Brachyury (B) transcript expression in day 3(D3), day 4 (D4), day 7 (D7), and day 14 (D14) hESC micromass culturessupplied with BMP2 at 48 hours of culture as determined by quantitativeReal Time RT-PCR. Expression levels were determined by the ΔΔCt methodusing GAPDH as endogenous control. Relative quantities were calculatedas −2^(ΔΔCt), and normalized to expression levels at day 2 which wereset to a value of 1. Values are the means of 3 (±SEM) (days 4, 7, and14) or 2 (±range) (day 3) determinations. (C-E) Expression levels ofBrachyury (C), Sox9 (D), and aggrecan (E) in day 3 (D3), day 4 (D4), andday 7 (D7) untreated control (BT−, blue), BMP2-treated (B+, red), BMP2plus TGFβ1-treated (BT+, green), and TGFβ1-treated (T+, purple) hESCmicromass cultures.

FIG. 8 is a photomicrograph of a section through a day 14 BMP2-treatedculture immunostained with an antibody against the interglobular domainof aggrecan. Cells which exhibit intracellular aggrecan staining arepresent throughout the extent of the culture. This indicates thatvirtually all of the cells that are surrounded by a type II collagenextracellular matrix (as shown in FIGS. 4A-C) are expressing thecartilage marker aggrecan as assayed by immunostaining with a cellautonomous marker.

FIGS. 9A-C are photomicrographs with a Table (Table 1) below FIG. 9C.FIG. 9A shows an Alcian blue and Nuclear Fast Red stained sagittalsection through day 14 hESC micromass cultures treated with BMP2. Cellssurrounded by an Alcian blue stainable extracellular matrix are presentthroughout virtually the entire extent of the section of theBMP2-treated culture, and, in addition, a small number of tubularstructures are present (arrows). FIG. 9B shows a section through an hESCmicromass culture treated with both BMP2 and TGF-β1 in which virtuallyall of the cells are surrounded by an Alcian blue cartilage matrix, andlittle, or no non-chondrogenic tissue is detectable. FIG. 9C shows asection through a BMP2+TGF-β1-treated hESC culture in which a smalltubule (arrow) is present in addition to the extensive cartilage tissue.Such tubules constitute only a very small percentage of theBMP2+TGF-β1-treated cultures, and the vast majority of the cultureundergoes cartilage differentiation. Table 1 shows that 88% of the cellspresent in the section of the day 14 BMP2-treated culture shown in FIG.9A are surrounded by an Alcian blue positive matrix. In the sections ofthe BMP2 and TGFβ1-treated cultures shown in FIGS. 9B and C, 97.3% ofthe cells are surrounded by Alcian blue-positive matrix. These resultsdemonstrate the substantial uniformity of chondrogenic differentiationby the BMP2 and BMP2/TGFβ1-treated hESC cultures in the method.

FIG. 10 is a photomicrograph showing an Alcian blue and Nuclear Fast Redstained sagittal section through a BMP2+TGF-β1-treated micromass cultureestablished from embryoid body (EB) cells. In addition to Alcianblue-stained chondrogenic tissue, a large amount of adipose tissue (ad)is present, as well tubules (arrows).

FIG. 11 is a photomicrograph demonstrating chondrogenic differentiationas assayed by Alcian blue staining of cartilage matrix of an iPS cellline derived from human foreskin fibroblasts subjected to the methodsdescribed herein. FIG. 11 (A) shows a day 14 control micromass cultureof iPSC maintained without BMP2 supplementation. Little Alcianblue-positive matrix is present. FIG. 11 (B) shows a day 14 BMP2 treatediPSC micromass culture. Intense and widespread Alcian blue stainingpresent throughout the extent of the culture.

FIGS. 12A-F are photomicrographs demonstrating the effect of iPSCdissociation method on chondrogenic differentiation in micromass cultureas assayed by Alcian blue staining. FIG. 12 (A-C) shows micromasscultures of BMP2-supplemented iPSC previously dissociated with trypsin(A), TrypLE Select (B) or Accutase (C) and stained whole-mount withAlcian blue after 7 days of culture. Comparable accumulation of Alcianblue is evident in each culture. FIG. 12 (D-F) shows iPSC culturesmaintained in the absence of BMP2 in which little Alcian blue-positivematrix is present regardless of the dissociation method used.

FIGS. 13A-C are photomicrographs demonstrating chondrogenicdifferentiation of iPSC micromass cultures in response to combined BMP2and TGFB1. FIG. 13A shows a day 14 micromass culture of iPSC maintainedin the presence of BMP2. FIG. 13B shows a day 14 iPSC micromass culturemaintained in the presence of a combination of BMP2 plus TGFβ1. FIG. 13Cshows a day 14 iPSC micromass culture maintained in the absence of BMP2or TGFβ1. Cultures supplemented with BMP2 or a combination of BMP2 andTGFβ1 accumulate comparable intense and widespread Alcian blue-positivematrix in contrast to iPSC micromass cultures which received no BMP2supplementation which accumulate little Alcian blue-positive matrix.

FIGS. 14A-D are photomicrographs demonstrating the mouse articularcartilage damage model. FIG. 14A and FIG. 14C show frontal sections of acontrol unoperated mouse knee. The medial meniscus (mm) is indicated.FIG. 14B and FIG. 14D show frontal sections of a mouse knee 8 weeksfollowing destabilization of the knee as a result of surgical ligamenttransection and partial meniscetomy. Absence of the medial meniscus isindicated by the asterisk. A localized region of articular cartilagedamage is present (arrow). FIGS. 14 C and D are higher magnificationviews of A and B.

FIGS. 15A-D are photomicrographs demonstrating the mouse digit tipregeneration model. FIG. 15A shows a control unoperated mouse digit tipin which bone is stained with Alizarin red. FIG. 15C shows a sectionthrough the control digit tip showing presence of soft and hard tissues.FIGS. 15B and 15D show a mouse digit tip amputated 6 weeks previously.All structures have undergone complete spontaneous regeneration.

FIGS. 16A-B are photomicrographs demonstrating derivation of iPSCs fromhuman fibroblasts from a patient with a genetic cartilage disorder(chondrodysplasia). FIG. 16A shows a living culture containing colonieswith appearance characteristic of undifferentiated pluripotent stemcells (10×). FIG. 16B shows immunocytochemical staining for markers ofpluripotent stem cells (SSEA-4 and Nanog) in the iPSCs.

FIG. 17 is a flow diagram showing the steps for producing chondrogeniccells from undifferentiated human embryonic stem cells or from inducedhuman pluripotent stem cells.

DETAILED DESCRIPTION OF THE INVENTION

Degenerative diseases of cartilage, e.g., osteoarthritis, are prevalentand debilitating chronic health problems and one of the main causes ofdecreased quality of life in adults (Magne D et al., 2005 Trends inMolecular Medicine, 11:519-526). Osteoarthritis (OA) is anon-inflammatory degenerative joint disease characterized by articularcartilage degradation and degeneration. OA affects most people over theage of 65, and it is estimated that 90% of the population over the ageof 40 exhibits some form of cartilage degeneration in their jointsresulting in pain and immobility (Song L et al., 2004 Cytotherapy,6:596-601). Treatment of degenerative cartilage diseases is a particularchallenge because of the limited capacity of the tissue for self-repairand renewal, making treatment of cartilage lesions a major clinicalproblem. Because of their unlimited capacity for self-renewal whilemaintaining the ability to differentiate into multiple cell types, humanembryonic stem cells (hESCs) derived from the inner cell mass of theblastocyst are a powerful tool for the repair of cartilage defects usingcell based tissue engineering therapies. Indeed, as described below,hESCs provide an unlimited supply of progenitor cells for cartilagerepair.

Using hESCs for cartilage repair requires efficiently directing theirdifferentiation into progenitor cells capable of participating in therepair of diseased cartilage. Prior to the invention, several culturesystems had been developed in which ESC-derived cells differentiate tovarious degrees into chondrocytes (; Elisseeff J et al., 2005 OrthodCraniofac Res, 8:150-161; Harkness L et al., 2009 Stem Cell Rev 5;353-368; Heng B C et al., 2004 Stem Cells, 22:1152-1167; Hoben G M et at2009 Stem Cells Dev 18:283-292; Hwang N S et al., 2006 Stem Cells,24:284-291; Hwang N S et al., 2006 Tissue Eng, 12:2695-2706; Jukes etal., 2008 Tissue Engineering Part A, 14:135-147; Kawaguchi J et al.,2005 Bone, 36:758-769; Koay E J et al., 2007 Stem Cells 25:2183-2190;Koay E J and Athanasiou K A 2009 tissue Eng Part A 15:2249-2257; KramerJ et al., 2000 Mechanisms of Development, 92:193-205; Kramer J et al.,2005 Cell Biology International, 29:139-146; Kramer J et al., 2005 AnatEmbryol, 210:175-185; Ofek G et al., 2009 Biomech Eng 131: 061011; Sui YP et al., 2003 Differentiation, 71:578-585; Toh W S et al., 2007 StemCells, 25:950-960; Toh W S et al., 2009 J Cell Mol Med 13B: 3570-3590;Vats A et al., 2006 Tissue Eng 12: 1687-1697; zur Nieden N I et al.,2005 BMC Dev Biol, 5:5-15). However, in these systems chondrocytesrepresented only a subpopulation of the cells that differentiate,complicating utilization of the cell population for cartilage repair.

Furthermore, most of the chondrogenic differentiation protocols utilizecells of embryoid bodies (EBs) derived from ESCs (Elisseeff J et al.,2005 Orthod Craniofac Res, 8:150-161; Harkness L et al., 2009 Stem CellRev 5: 353-368; Heng B C et al., 2004 Stem Cells, 22:1152-1167; Hoben GM et al 2009 Stem Cells Dev 18:283-292; Hwang N S et al., 2006 StemCells, 24:284-291; Hwang N S et al., 2006 Tissue Eng, 12:2695-2706;Jukes et al., 2008 Tissue Engineering Part A, 14:135-147; Kawaguchi J etal., 2005 Bone, 36:758-769; Koay E J et al., 2007 Stem Cells25:2183-2190; Koay E J and Athanasiou K A 2009 tissue Eng Part A15:2249-2257; Kramer J et al., 2000 Mechanisms of Development,92:193-205; Kramer J et al., 2005 Cell Biology International,29:139-146; Kramer J et al., 2005 Anat Embryol, 210:175-185; Ofek G etal., 2009 J Biomech Eng 131: 061011; Sui Y P et al., 2003Differentiation, 71:578-585; Toh W S et al., 2007 Stem Cells,25:950-960; Toh W S et al., 2009 J Cell Mol Med 13B: 3570-3590; Vats Aet al., 2006 Tissue Eng 12: 1687-1697; zur Nieden N I et al., 2005 BMCDev Biol, 5:5-15; Waese and Stanford 2010 Stem Cell Res September 6).EBs are three-dimensional aggregates of cells formed by incubatingsuspensions of undifferentiated ESCs or colonies on non-adhesivesubstrates. The cells of EBs can differentiate into semi-organizedtissues composed of cell types from all three germ layers of the embryo.The cellular environments and interactions that occur in EBs aredifficult to precisely control, fostering cellular heterogeneity. Thus,utilizing EB-derived cells hinders the ability to obtain homogeneouspopulations of chondrogenic cells that can be used for tissue repair.

Some protocols have been reported in which the chondrogenicdifferentiation of hESCs without prior EB formation have been attempted.One protocol involved co-culturing the hESCs for an extensive period(several weeks) with differentiated bovine articular chondrocytes (HwangN S et al., 2008 PLoS ONE, 3:1-10). Another protocol involvedpre-culturing the ESCs in ill-defined conditioned medium from ahepatocarcinoma cell line (Hwang Y S et al., 2008 Stem Cells Dev,17:971-978) however this study resulted in limited and non-uniformchondrogenic differentiation (Hwang N S et al., 2008 PLoS ONE, 3:1-10).

Another study has utilized micromass cultures of mouse ES cells toattempt to obtain cartilage differentiation without prior EB formation(Yamashita A et al., 2009, Cell Death and Differentiation 16:278-286).The Yamashita protocol involved adding growth factors at the start ofculture, and resulted in considerable cell death and detachment from theculture substrate. Moreover, the cultures exhibit only patchy and nonuniform Alcian blue staining and little or no expression of thedefinitive cartilage marker aggrecan detectable by RT-PCR. The cultureswere reported to express the hypertrophic cartilage marker collagen type10 and undergo mineralization, indicating the cells in the culture haveundergone hypertrophic chondrocyte maturation very early in the cultureperiod. These characteristics represent a significant drawback to themethod, because hypertrophic chondrocyte maturation is associated withosteoarthritis. The cultures were also reported to express the bloodvessel marker flk1, indicating the heterogeneity of the culture. Therelatively small amount of chondrogenic tissue that forms undergoeshypertrophic maturation, making it inappropriate for cartilage repair.Indeed a second study by the same authors (Yamashita A et al., 2010 PLoSOne 5:e10998) reported that the cultures underwent further hypertrophyand ultimately formed bone-like tissue in vitro.

In contrast, the methods described herein yield substantially uniformchondrogenic differentiation as assayed by histological and molecularanalyses. The methods yield cells that exhibit high level expression ofaggrecan and other cartilage markers. The progressive differentiationthat these cultures undergo enables us to obtain relatively purepopulations of cells at discrete stages of differentiation that may beused for cartilage repair. Furthermore the cells do not undergohypertrophic maturation, making them particularly attractive forcartilage repair.

These differences relate, in part, to the timing of growth factoraddition. Yamashita added growth factors at the start of high densityculture (zero time), whereas in the method described herein, thecultures receive growth factor after the 24 or 48 hours afterestablishment of high density culture. The timing of growth factorsupplementation is critical for chondrogenic differentiation, astreatment of chondrogenic progenitors in micromass culture with BMP atthe start of culture (time 0) causes cell death and inhibitschondrogenic differentiation (Fisher et al 2007) and others have shownthat treatment of chondrogenic progenitors in vivo with BMP causesmassive cell death (Macias et al 1997). The cultures also receive ROCKinhibitor to promote hESC survival (Watanabe K et al., 2007; Li X 2009).Suitable inhibitors include Y27632 and H1152 (both available fromCalbiochem), AR-12286 (Aerie Pharmaceuticals), as well as HA-1100.HCl([Hydroxyfasudil; 1-Hydroxy-5-isoquinolinesulfonyl)homo-piperazine],3-(4-Pyridyl)-1H-indole, H-1152.2HCl ([H-1152P;(S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine,and N-(4-Pyridyl)-N′-(2,4,6-trichlorophenyl) urea (the latter four ofwhich are available from Alexis Biotchemicals).

Protocols for indirect differentiation of hESC into chondrocytesutilizing a mesenchymal stem cell-like intermediate have also beenreported. However, several of these protocols utilize EBs to produce themesenchymal stem cell-like intermediate (Lee E J et at, 2010 Tissue EngA 16:705-717; Brown S E et al., 2008, Cells Tiss Org 189:256-260;Mahmood A et al., 2010 J Bone MM Res 25: 1216-1233; Hwang et al 2008;Cohen S et al., 2010 Tissue Eng A 16: 3119-3139; Harkness L et al., 2009Stem Cell Rev 5: 353-368) which as discussed above, introduces cellularheterogeneity. Similarly, an EB step is used in a protocol reportingchondrogenic differentiation from a mesenchymal stem cell intermediatederived from human stem cells isolated from human gonadal ridge (hEGcells, Varghese S et al., 2010 Stem Cells 28: 765-774). Other protocolsrequired co-culture with OP9 cells, a line of bone marrow derived mousestromal cells (Barberi T et al., 2005 PLoS Med 2 e161) or coculture withhuman articular chondrocytes (Bigdeli N et al., 2009 Stem Cells27:1812-1821) in order to produce the mesenchymal intermediate. Severalprotocols obtained the mesenchymal stem cell-like intermediate cellpopulation through use of FACS sorting to identify hESC subpopulationsexpressing mesenchymal stem cell markers (Lian Q et al., 2007 Stem Cells25:425-436; Kopher R A et al., 2010 Bone 47:718-728; Stavropoulos M E etat, 2009 Curr Prot Stem Cell Biol 9:1F.8.1-1F.8.10; Barberi T et al.,2005 PLoS Med 2 e161; Barberi T et al., 2007 Nature Med 13: 642-651) orthrough repeated passaging and extensive long term (several weeks)culture of the hESC at low density (Nakagawa T et al., 2009 ArthritisRheum 60: 3686-3692; Arpornmeaklong P et at, 2009 Stem Cells Dev18:955-968; Boyd N L et at, 2009 Tissue Eng A 15:1897-1908) in order toproduce the spindle-shaped, fibroblast/mesenchymal-like cells which arethen subjected to various chondrogenic differentiation protocols.However, in these protocols, chondrogenic differentiation obtained wasnon-uniform, as indicated by patchy immunostaining for collagen type II(Arpommeaklong P et at, 2009 Stem Cells Dev 18:955-968); lack ofhomogeneity of the tissue (Kopher et al 2010), non-uniform type IIcollagen and sox9 distribution (Barberi T et al., 2007 Nature Med 13:642-651; Stavropoulos M E et at, 2009 Curr Prot Stem Cell Biol9:1F.8.1-1F.8.10) formation of non-uniform, tissue with little Alcianblue staining (Lian Q et al., 2007 Stem Cells 25:425-436); andproduction of a poorly-characterized mass that detached from the cultureplate (Boyd N L et al., 2009 Tissue Eng A 15:1897-1908). A protocolwhich subjected the hESC-derived mesenchymal fibroblast-like cells topellet culture in the presence of BMP7 (Nakagawa T et al., 2009Arthritis Rheum 60: 3686-3692) also produced non-uniform chondrogenicdifferentiation with the cultures exhibiting an extensiveundifferentiated central core with chondrogenic differentiation beinglimited to cells at the periphery of the pellet. Chondrogenicdifferentiation in pellets treated with both BMP7 and TGFβ1 (Nakagawa Tet at, 2009 Arthritis Rheum 60: 3686-3692) also was not uniform, with anundifferentiated central core and overall weak collagen type IIimmunostaining at the periphery. The expression of the definitivecartilage marker aggrecan by these cultures was not significantlyincreased relative to untreated control pellets.

The results of these earlier methods are in contrast to chondrogenicdifferentiation by hESC in methods described herein, which yield direct,progressive and substantially uniform cells. Another advantage is thatthe methods of the invention yield cells that are synchronized atdiscrete stages of differentiation. For example, cells are harvested atdiscrete points to obtain chondroprogenitors (2-3 day cells,characterized as just entering the cartilage lineage), earlychondrocytes (day 4 cells, characterized as being in an early phase ofchondrogenic differentiation), or fully differentiated chondrocytes(7-14 day cells have uniformly undergone overt differentiation intochondrocytes but have not undergone hypertrophic maturation).

Days of culture after high density culture Cell population establishmentMarker profile by qRT-PCR Chondroprogenitors 2-3 Moderate Brachyury,Sox9 and Col2a1. Low aggrecan. Early chondrocytes 4 High Brachyury andSox9, moderate aggrecan and Col2a1 Fully differentiated 7-14 LowBrachyury and Sox9, chondrocytes high aggrecan and Col2a1. Col10a1 andOPN not detectable.Each of the cell populations described above are ideally suited fortreatment of specific clinical conditions. For example,chondroprogentors (day 2-3 cells) are preferentially used for limb orfinger regeneration, mensical or ligament repair, and fracture repair.Early chondrocytes (day 4 cells) are used for repair of articularcartilage defects due to injury, for repair or prevention of articularcartilage due to chronic degenerative disease (OA or RA), forregeneration and replacement of lost cartilage tissue due to trauma orcongenital defect, for meniscal or fracture repair, and for repair ofthe fibrocartilage and cartilage of the temporomandibular joint. Fullydifferentiated chondrocytes are used for articular cartilage repairfollowing acute or traumatic injury, or for repair or prevention ofarticular cartilage damage due to chronic OA or RA, as well as forfracture repair via endochondral ossification. Each stage is useful formodeling of cartilage development and differentiation, and iPSC derivedcells in particular are useful for patient specific repair andregeneration, as well as for modeling of human disease, design andtesting of targeted therapeutics, and for disease-specific repair ofgenetically compromised cartilage tissue.

Chondrogenic differentiation of iPSC has been reported (Medvedev S P etal., 2010 Stem Cells Dev October 17; Lian Q et al., 2010 Circulation121:1113-1123). However, one protocol involved formation of EBs from theiPSC (Medvedev S P et al., 2010 Stem Cells Dev October 17); and theother protocol required sorting of the iPSC by fluorescence-activatedcell sorting (FACS) to identify subpopulations expressing mesenchymalstem cell markers in order to obtain a mesenchymal stem cell-likeintermediate cell population prior to induction of chondrogenicdifferentiation (Lian Q et al., 2010 Circulation 121:1113-1123).Chondrogenic differentiation in this protocol resulted in only about 60%of the cells being immunoreactive for collagen type II (Lian Q et al.,2010 Circulation 121:1113-1123). Yet another advantage of the presentmethods is the avoidance of the use of FACS (which is time-consuming andmay introduce a risk of contamination).

The method described herein does not utilize EBs. The method does notinvolve coculture of hESCs or iPSCs with differentiated bovine articularchondrocytes, human articular chondrocytes, or mouse OP9 cells. Themethod does not involve pre- or co-culture with medium fromhepatocarcinoma cells. Nor do the methods involve long-term passagingand culture in monolayer, or cell sorting via FACS. The methods arefaster (less than 3 weeks, less than 2 weeks, or less than 1 week, e.g.,cells enter chondrogenic lineages in as little as 2-3 days afterestablishment of high density cell cultures). The present method resultsin progressive, substantially uniform chondrogenic differentiation whichenables production of populations of cells at discrete stages ofdifferentiation for cartilage repair and other uses.

Described below are culture systems and conditions of the invention thatpromote the rapid, direct, progressive, and uniform differentiation ofundifferentiated pluripotent hESCs into the chondrogenic lineage withoutprior embryoid body (EB) formation. Undifferentiated pluripotent hESCsand cells of embryoid bodies (EBs) derived from hESCs were subjected tothe high density micromass culture conditions to direct thedifferentiation of embryonic limb bud mesenchymal cells intochondrocytes (Gay SW and Kosher R A, 1984 J Exp Zool, 232:317-326;Kosher R A et al., 1986 Dev Biol, 118; 112-117; Kosher R A et al., 1986J Cell Biol, 102:1151-1156; Kulyk W M et al., 1991 Matrix, 11:282-288).The high density micromass culture system simulates the closejuxtaposition of cells and cellular interactions that characterize theonset of the chondrogenic differentiation of mesenchymal progenitorcells in the developing embryonic limb. Micromass cultures ofundifferentiated pluripotent hESCs treated with BMP2 alone (or thecombination of BMP2 and TGFβ3 administered together) directly undergoprogressive and substantially uniform (e.g., at least 88%)differentiation into the chondrogenic lineage without prior EBformation.

As an alternate approach to supplementation with BMP2 and TGFβ1,cultures are supplemented with other growth factors includingbone-morphogenetic protein-4 (BMP4, NG_(—)009215 (GI:219521814),incorporated herein by reference); bone morphogenetic protein-7 (BMP7,NM_(—)001719 (GI:187608319), incorporated herein by reference); growthand differentiation factor-5 (GDF5, NG_(—)008076 (GI:193083169),incorporated herein by reference); transforming growth factor beta-3(TGFβ3, NG_(—)011715 (GI:225735563), incorporated herein by reference);or insulin like growth factor-1 (IGF-I, NG_(—)011713 (GI:225735562),incorporated herein by reference). Each of these factors promotedifferentiation into the chondrogenic lineage (Waese E Y and Stanford WL 2010 Stem Cell Res September 6; Nakagawa T et al., 2009 ArthritisRheum 60: 3686-3692; An C et al., 2010 Ann Biomed Eng 38: 1647-1654;Moore Y R et al., 2010 J Clin Periodontol 37: 288-298).

As an alternate approach to micromass culture, hESC are subjected topellet culture. In this method, dissociated hESC or iPSC (approximately1×10⁵-1×10⁷ cells) are pelleted in a microfuge tube. The pellet is thensubjected to the same chondrogenic differentiation protocols asdescribed above for micromass culture. Growth factor (s) are added tothe pellet and diffuse into the pellet to induce differentiation of thecells. The pellet itself is then used for implantation, infusion orother means of administration to a joint or joint space or other repairtissue for therapy. This protocol also achieves direct, progressive andsubstantially uniform chondrogenic differentiation of hESC without EBformation. The pellet culture protocol is also used to direct thedifferentiation of iPSC into the chondrogenic lineage.

Cell Culture Systems: System I and System II

The cells are prepared using one of two systems. In system I, highdensity cultures are established and cultured for 24 hours at whichpoint chondrogenic medium containing BMP2 or the combination of BMP2 andTGFβ is added to the cells. In system II, high density cultures areestablished and cultured for 24 hours at which point chondrogenic mediumwithout growth factor(s) is added to the cells. At the beginning of the3^(rd) day, BMP2 or the combination of BMP2 and TGFβ is added to thecells. One day later (at the end of day 3), the cells substantiallyuniformly enter the chondrogenic lineage as evidenced by gene expressionprofiles and physical characteristics/histology. A flow chartillustrating the timing of culture, growth factor addition, and stage ofdifferentiation is shown in FIG. 17.

As described below, the gene expression profiles of hESC-derived cellsharvested at various times during the progression of theirdifferentiation enabled the identification of cells in different phasesof the chondrogenic lineage ranging from cells just entering thechondrogenic lineage to overtly differentiated chondrocytes. The methodsdescribed herein enable analysis of the ability of hESC-derivedprogenitor cells in different phases of chondrogenic lineage to repaircartilage using cell-based tissue engineering therapies. The uniform andprogressive chondrogenic differentiation that occurs also facilitatesthe identification of genes, signals, and regulatory networks involvedin the progressive conversion of undifferentiated pluripotent hESCs intoprogenitor cells determined to differentiate into the chondrogeniclineage.

Methods of Therapy

Approaches for administering the cells include direct implantation ofthe micromass or pellet cultures into the damaged area via surgicalmeans, such as open knee surgery as for autologous chondrocyteimplantation (ACI, Zazlav K et al., 2009 Am J Sports Med, 37:42-55) orarthroscopically in a scaffold-free approach (Jubel A et al., 2008 Am JSports Med 36: 1555-1564; Erggelet C et al 2003 Arthroscopy 19:108-110). In some procedures, the cultures are also dissociated bycollagenase digestion or other means, and cells seeded into scaffolds orother supports. Such supports include collagen membranes (e.g.Chondrogide), hyaluronan-containing matrices (e.g. Hyaff-11),polyglactin fleece or other matrices. The cell-seeded matrices are theneither surgically implanted (Kon et al., 2009 Am J Sports Med 37:156S-166S) as for matrix-induced autologous chondrocyte repair (MACI)(Brittberg, 2010 Am J Sports Med, 38: 1259-1271) or HyalgraftC (Gobbi Aet al., 2006: Am J Sports Med 34: 1763-1773); or implanted viaarthroscopy (Gianni E et al., 2008 Am J Sports Med 36:873-880; Nixon A J2002 Clinical Tech Equine Practice 1: 257-269). The dissociated cellsare also encapsulated in collagen, chitosan, agarose orhyaluronan-containing gels and the cell-gel mixture used to fill thedamaged region in a surgical procedure (Funayama A et al., 2008 J OrthopSurg 13: 225-232; Hoemann C et al., 2005 OsteoArthritis Cart 13:318-329; Emans P J et al., 2010 PNAS 107:3418-3423). In these methods,the concentration of cells to be used is between (1×10⁶-2×10⁷ cells/ml).

Alternately, the dissociated cells are injected or infused into the kneevia direct intra-articular injection in a non-invasive manner. Thenumber of cells to be introduced is between (1×10⁶-2×10⁷ cells/ml). Thecells are injected in saline as for introduction of autologousmesenchymal stem cells (MSC) into the injured or arthritic knee (CentenoC J et al., 2008 Pain Physician 11: 343-353) or injection of MSC intojoints of rats, pigs and goats with cartilage damage (Horie M et al.,2009 Stem Cells 27:878-887; Lee K B et al., 2007 Stem Cells25:2964-2971; Murphy J M et al., 2003 Arthritis Rheum 48:3464-3474). Inanother approach, cells are injected in conjunction with othersubstances including hyaluronan preparations used forviscosupplementation (e.g. Synvisc, Supartz) which alleviate clinicalarthritis pain and immmobility (Petrella R J and Petrella M 2006, JRheumatol 33: 951-956; Brander V A and Stadler T S 2009 Phys Sports Med37: 38-48); or in conjunction with growth factors such as BMP7 whichdelay osteoarthritic progression (Hunter D J et al., 2010, BMCMusculoskelet Disord 11: 232; Hayashi M et al., 2010 J Orthop Res 28:1502-1506).

One approach to cell delivery is arthroscopic administration. In somecases, the cells are administered or implanted before, after or during asurgical or arthroscopic procedure to repair an associated defect orcondition, e.g., cells are administered to a joint space in conjunctionwith an ACL repair procedure, meniscus repair, rotator cuff repair, orother procedure. This approach has been used to evaluate repair ofmeniscal injury by mesenchymal stem cells in animals (Horie M et al.,2009 Stem Cells 27:878-887) and is suitable for humans (Centano C J etal., 2008 Med Hypotheses 71:900-908.)

The cells and methods are useful to assist in repair of sports-related,combat-related and other traumatic injuries to bones, cartilage andlimbs (Goldstein 2006 J Am Acad Orthop Surg 14: S152-156; Sundelacruz Sand Kaplan D L 2009 Sem Cell Dev Biol 20: 646-655). The cells are usefulfor repair of long bone fractures, which repair via endochondralossification, a process which recapitulates the normal developmentalsequence involving ossification on a cartilage template (Kronenberg H M2003 Nature 423: 332-336). Cells are introduced into the region of thefracture by injection or implantation, and undergo remodeling to formbone for repair, as described in methods utilizing mesenchymal stemcells (Kallai I et al., 2010 J Biomech 43: 2315-2320; Scotti C et al.,2009 PNAS 107: 7251-7256).

The cells are also useful in methods of regenerating fingers or limbslost to traumatic injury due to accident or military conflict, orcongenital defect. Lower animals including newts and frogs possess theability to regenerate limbs, but this ability is largely lost in adultmammals (Muller T L et al., 1999 Sem Cell Dev Biol 10: 405-413) due toinsufficient cells and signals required to carry out a regenerationresponse (Gurtner G C et al., 2007 Ann Rev Med 58:299-312; Muneoka K etal., 2008 Sci Am 298:56-63).

A murine model of limb regeneration was established. In this model,distally amputated digits undergo a spontaneous regeneration response,whereas proximally amputated digits fail to regenerate (Han Metal., 2008Dev Biol 315: 125-135). This model provides a system for testing theability of hESC and iPSC derived chondroprogenitor cells, particularlycells just entering the skeletal and chondrogenic lineage (e.g. day 2,day 3 or day 4 cells) to participate in or promote limb regeneration ina mammal. Methods of delivery for therapy for regeneration of limbs(arms or legs, hands or feet) or digits (fingers or toes) includeintroduction of the cells produced by the method into the digit or limbby injection or implantation. Cells are introduced in scaffolds or gelswith or without growth factors. The digit regeneration model isdescribed in FIGS. 15A-D.

Disease Models

iPSC obtained from patients with genetic diseases are used for in vitromodeling of human disease and as a system for design and testing oftargeted therapeutics (Amabile G and Meissner A 2009 Trends Molec Med15: 59-68; Laustriat D et al., 2010 Biochem Soc Trans 38: 1051-1057).The invention is also useful for in vitro modeling of genetic cartilagediseases and for design and testing of therapeutics. These disorderswhich include chondrodysplasia and achondoplasia cause disfiguring anddisabling short stature and even death (Krakow D and Rimoin D 2010 GenetMed 12: 327-341). For this purpose, iPSC are derived from fibroblasts orother cell types obtained from individuals with genetic disordersaffecting cartilage (e.g., chondrodysplasia, achondroplasia or others)and the method described herein is applied to induce chondrogenicdifferentiation. The iPSC derived chondrogenic cells recapitulate thedisease process in vitro, enabling mechanistic study of diseasepathology and design and testing of targeted therapeutics. The approachextends to chondrodysplasias in which mechanisms are largely unknown(Krakow D and Rimoin D 2010 Genet Med 12: 327-341), such as those causedby defects in extracellular matrix protein synthesis or sulfation;metabolic enzymes, ion channels or transporters; macromolecular folding,processing or degradation; hormones, growth factors, receptors, orsignal transducers; transcription factors; RNA processing; orcytoskeletal proteins. The approach is applied to gene profiling toidentify new genes important in normal or abnormal cartilage function ornew targets for therapy (Pogue R et al., 2004 Matrix Biol 23: 299-307).The approach may be combined with gene targeting to attempt patientspecific cell-mediated therapy of mongenic chondrodysplasias (Wong G Kand Chiu A T 2010 Biotechnol Adv July 24).

Experimental models in large and small animals are used to evaluatecartilage repair (Chu C R et al., 2010 Tissue Eng Part B 16: 105-115).In one model, the knee joint is de-stabilized by surgical disruption ofthe knee ligaments and/or partial meniscetomy, which results ininappropriate focused weight bearing and causes localized articularcartilage destruction. The cartilage defect produced in this model ishighly reproducible and the onset and progression of cartilagedestruction faithfully replicates what is seen in chronic humanosteoarthritis. This model has been used to evaluate cell-mediatedrepair of damaged articular cartilage by mesenchymal stem cells injectedinto the joint of various animals (e.g. Murphy J M et al., 2003Arthritis Rheum 48:3464-3474; Kubo S et al., 2009 Arthritis Rheum 60:155-165). This model has been extended to rodents (Kamekura S et al.,2005 Osteoarthritis Cart 13: 632-641; Glasson S S et al., 2007Osteoarthritis Cart 15:1061-1069; Welch I D et al., 2009 Arthritis ResTher 11:R14). This model has now been established in immune-compromisedmice for use in testing in vivo repair by the hESC and iPSC derivedchondrogenic cells (FIGS. 14A-D).

Another model used to evaluate cartilage repair utilizes surgicallygenerated articular cartilage defects (Ahern B J et al., 2009Osteoarthritis Cart 17: 705-713). In this model the non-loading regionof the patellar groove of the femur is exposed, and a full-thicknessdefect is mechanically created by carving a groove or punching out ahole extending from the cartilage surface to the subchondral bone. Thisapproach is used for large animals and has also been adapted to the rat(Dausse Y et al., 2003 Osteoarthrtis Cart 11: 16-28) and mouse (EltawilN M et al., 2009 Osteoarthritis Cart 17:695-704; Osteoarthritis Cart 6:695-704). This model simulates articular cartilage damage followingacute injury, and as in humans, failure to heal the defect leads tofurther cartilage degeneration outside the damaged region and overt OA.

Example 1 Culture of hESCs and Embryoid Body Formation

The H9 hESC line generated at the WiCell Research Institute (Thomson J Aet al., 1998 Science, 282:1145-1147). The H9 cells were cultured in theserum-free hESC medium previously described (Toh W S et al., 2007 StemCells, 25:950-960) on a feeder layer of irradiated CF1 mouse embryonicfibroblasts (MEFs) (see, FIG. 1A) prepared using standard protocols bythe University of Connecticut Stem Cell Core. Colonies were passagedevery 4-6 days after treatment with 1 mg/ml collagenase IV (Invitrogen).hESCs from passage numbers 36 to 45 were used in these studies.

To generate EBs, hESC colonies were detached from the MEF feeder layersby treatment with 0.1% dispase (Invitrogen) for 20-30 minutes at 37° C.,suspended in EB formation medium (Toh W S et al., 2007 Stem Cells,25:950-960) in T75 Ultra-Low Attachment Flasks (Corning, Lowell, Mass.),and incubated for 5 days. Media was changed every 48 hours. Thesuspended hESC colonies form EBs consisting of three-dimensionalaggregates of cells as shown in FIG. 1B.

Preparation of Micromass Cultures from Eb Cells and fromUndifferentiated Hescs

Micromass cultures were prepared from dissociated cells of both day 5EBs (FIG. 1B) and undifferentiated hESC colonies (FIG. 1A). Prior todissociation, hESC colonies were detached from the MEF feeder layer asdescribed above. The detached hESC colonies and EBs were dissociatedinto single cells with 0.05% trypsin/EDTA (Invitrogen), followed bypassage of the cell suspension through a 40 μm cell strainer (BDBiosciences, Franklin lakes, NJ). The dissociated cells were suspendedat 2×10⁷ cells/ml in high-glucose DMEM supplemented with 10% FBS (FBS,Hyclone) and 10% KSR. Micromass cultures were prepared by spotting 10 μlof the cell suspensions (2×10⁵ cells) in each well of 24-well tissueculture dishes (Nunc; Fisher). After a two hour incubation at 37° C. ina humidified 5% CO₂ incubator to facilitate cell attachment, 0.5 ml ofthe same medium was added to each well. After 24 hours (the beginning ofday 2 of culture), the medium was removed and the cultures supplied withthe serum-free chondrogenic medium (Toh W S et al., 2007 Stem Cells,25:950-960). Chondrogenic medium contains high-glucose DMEM supplementedwith ITS (6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 μg/mlselenium), 1.25 mg/ml bovine serum albumin, 5.35 μg/ml linoleic acid (BDBiosciences), 1% KSR, 40 μg/mL-proline (Sigma-Aldrich), 50 μg/mlascorbic acid 2-phosphate (Sigma-Aldrich), 1% nonessential amino acids(Invitrogen), 10-7 M dexamethasone (Sigma-Aldrich), and 100 units/100 μgpenicillin/streptomycin (Invitrogen). The cells are then cultured in thepresence or absence of either 100 ng/ml recombinant human BMP2 (R&DSystems, Minneapolis), 10 ng/ml of recombinant human TGF-β1 (R&DSystems, Minneapolis), or a combination of 100 ng/ml of recombinanthuman BMP2 plus 10 ng/ml of recombinant human TGF-β1. In someexperiments, hESC micromass cultures were cultured for 24 hours(throughout day 2) in chondrogenic medium lacking growth factors, afterwhich (at the beginning of day 3 of culture) they were supplied withfresh chondrogenic medium supplemented with growth factors as describedabove. Media including growth factors was changed every 48 hoursthroughout the culture period.

Alcian Blue Staining, Type II Collagen and Aggrecan Immunostaining

The accumulation of cartilage matrix was monitored histochemically bystaining micromass cultures with Alcian blue, pH 1.0 as previouslydescribed (Gay S W and Kosher R A, 1984 J Exp Zool, 232:317-326). Forimmunostaining of type II collagen, micromass cultures were fixed for 30minutes in 4% paraformaldehyde in phosphate buffered saline (PBS),washed in PBS, scraped off the tissue culture plate, dehydrated,embedded in paraffin, and sectioned sagitally at 7 μM.Immunohistochemistry was performed using a Vectastain Elite ABC kit(Vector, Burlingame, Calif.) and a monoclonal antibody against type IIcollagen (anti-collagen Type II, clone 6B3; Chemicon) and a monoclonalantibody against the interglobular domain of the cartilage marker,aggrecan (6-B-4; Abeam). Sections were de-paraffinized, quenched in 0.5%H₂O₂ in methanol for 15 minutes, and antigen retrieval was performed byincubation with pepsin (Labvision). Sections were blocked with 5% normalhorse serum in TBS, and then incubated for 1 hour with the type IIcollagen antibody diluted 1:200. The sections were then incubated for 1hour at room temperature with biotinylated horse anti-mouse IgG diluted1:200, treated with an avidin-biotin horseradish peroxidase complex(Vectastain ABC kit), and developed with diaminobenzidine/H₂O₂ (Vector).Negative controls were not incubated with the primary antibody, andexhibited little or no staining.

Real Time Reverse Transcription-Polymerase Chain Reaction (Real TimeRT-PCR)

Total RNA was extracted from micromass cultures using the Qiagen RNeasyMini kit (Qiagen, Chatsworth, Calif.), and treated with RNase-free DNase(Ambion, Austin, Tex.) to eliminate possible genomic DNA contamination.Two μg of RNA per 20 μl of reaction volume were reverse transcribed intocDNA using the High Capacity cDNA Reverse Transcription Kit (AppliedBiosystems, Foster City, Calif.). Real-time RT-PCR was performed usingthe ABI Prism 7900 Sequence Detection System, TaqMan Gene ExpressionMaster Mix, and TaqMan Gene Expression Assays (Applied Biosystems) forSox9 (assay ID Hs00165814_m1), aggrecan (assay ID Hs00153936_m1), typeII collagen (Col2a1) (assay ID Hs00264051_m1), type X collagen (Col10a1)(assay ID Hs00166657_m1), osteopontin (opn) (assay ID Hs00960942_m1),Indian hedgehog (Ihh) (assay ID Hs01081801_m1), Brachyury (assay IDHs00610080_m1), and glyeraldehyde-3-phosphate dehydrogenase) (GAPDH)(Hs99999905_m1). Gene expression levels were determined by the ΔΔCtmethod using GAPDH as the internal reference gene. Relative quantitiesof each gene were calculated as −2^(ΔΔCt) in duplicate samples preparedfrom each of 2-3 independent micromass cultures for each treatment andtime point analyzed. Thermal cycling conditions were 50° C. for 2minutes, 95° C. for 10 minutes followed by 40 cycles of 15 seconddenaturation at 94° C. and 1 minute extension at 60° C.

Example 2 Direct and Progressive Chondrogenic Differentiation ofUndifferentiated Pluripotent hESCs

In view of the ill-defined cellular environments and inherent cellularheterogeneity characteristic of EBs, the ability of undifferentiatedpluripotent H9 hESCs (FIG. 1A) to directly differentiate into thechondrogenic lineage when subjected to high density culture wasexamined. High density cultures are characterized by a concentration ofgreater than 1×10⁵ cells per 10 μl of medium. For example, theconcentration of cells is in the range of 1-4×10⁵ cells per 10 μl ofmedium, e.g., 2×10⁵ cells per 10 μl. The volumes are scaled up asdesired for larger scale cultures provided that the cell density/ratioremains in the range described above.

One day after their establishment, micromass cultures of dissociated H9cells were supplied with serum-free “chondrogenic medium” in thepresence and absence of 100 ng/ml of BMP2. As shown in FIG. 4A, only afew small Alcian blue staining areas were present in day 14 untreatedmicromass cultures established directly from dissociatedundifferentiated H9 hESCs cells. In the presence of exogenous BMP2,virtually uniform intensely staining Alcian blue-positive matrixaccumulates in the day 14 BMP2-treated hESC micromass cultures (FIG.4B). In addition to uniform Alcian blue staining,cartilage-characteristic type II collagen detectable by immunostainingwith a type II collagen antibody was present throughout the extent ofBMP2-treated cultures (FIG. 4C).

The progression of accumulation of Alcian blue matrix by micromasscultures of undifferentiated H9 cells cultured as described above isshown in FIG. 5A-C. Little Alcian blue staining was detectable after 3days of culture (FIG. 5A), but by day 7 relatively uniform Alcian bluestaining was detectable throughout the culture (FIG. 5B). At day 14,more intensely staining Alcian blue-positive matrix was presentthroughout the entirety of the culture (FIG. 5C). These results indicatethat undifferentiated pluripotent hESCs directly and quite uniformlyundergo chondrogenesis when provided with the appropriate cellularenvironment (high density micromass culture) and exogenous signalingmolecules such as BMP2 without the necessity of passing through an EBstage.

A modification of the chondrogenic differentiation protocol describedabove further enhances the progression of the differentiation of hESCsinto the chondrogenic lineage. In this modified protocol, one day afterbeing established, the hESC micromass cultures are supplied withserum-free “chondrogenic medium” lacking BMP2, and then 24 hours latersupplied with 100 ng/ml of BMP2. As shown in FIG. 5D-F, although littleAlcian blue positive matrix was detectable in day 3 cultures, uniformand fairly intense Alcian blue matrix was present throughout the extentof day 7 cultures. By day 14, a very intensely staining Alcian bluematrix was present throughout the entirety of the cultures (FIG. 5F).The extent and intensity of Alcian blue staining was greater utilizingthis modified protocol in which exogenous BMP2 was provided on day 3(FIGS. 5D-F) than in the protocol in which the hESC cultures weresupplemented BMP2 on day 2 (FIG. 5A-C).

Example 3 Characterization of Cells in Various Phases of theChondrogenic Lineage in hESC Micromass Cultures by Gene ExpressionProfiling

To confirm and quantify the progression of the differentiation ofpluripotent hESCs into the chondrogenic lineage and to furthercharacterize progenitor cells in different phases of the lineage, theexpression of marker genes characteristic of different stages of thechondrogenic lineage was examined by quantitative Real Time RT-PCR. Geneexpression patterns were analyzed at various times in BMP2-treatedmicromass cultures of pluripotent hESCs subjected to the modifiedchondrogenic differentiation protocol that leads to the progressivechondrogenic differentiation assayed by Alcian blue matrix staining asshown in FIGS. 5D-F above.

As shown in FIG. 7A, at the end of day 3 of culture, which is 24 hoursafter the addition of exogenous BMP2, there is a striking 7-foldupregulation in the expression of the chondrogenic transcription factorSox9. Sox9 has been characterized as a “master regulatory gene” forcartilage differentiation, and is a critical regulator of virtually ofall the early phases of chondrogenesis (Akiyama H et al., 2002 Genes andDevelopment 16:2813-2828; Lefebvre V and Smits P, 2005 Birth Defects ResC Embryo Today, 75:200-212). Thus, the striking upregulation of Sox9expression in day 3 cultures is consistent with the entrance of thecells into the chondrogenic lineage. It is also noteworthy that the day3 cultures exhibit a 24-fold upregulation in the expression of thetranscription factor Brachyury (FIG. 7B). Brachyury is a marker of themesodermal lineage and is also expressed by the prechondrogenicmesodermal cells that will give rise to cartilage in the developing limb(Herrmann B G, 1995 Seminars in Developmental Biology, 6:385-394;Hoffmann A et al., 2002 J Cell Sci, 115:769-781; Liu C et al., 2003Development, 130:1327-1337). Thus, the upregulated expression ofBrachyury along with Sox9 further indicates that day 3 cultures haveentered into the chondrogenic lineage. Although day 3 cultures exhibitupregulated expression of Sox9 and Brachyury, the cultures do notexhibit upregulated expression of the cartilage marker aggrecan (FIG.7A). In day 3 cultures, aggrecan is expressed at the same low basallevel as it is at day 2. Aggrecan is the major sulfated proteoglycan ofcartilage matrix and is a definitive highly specific molecular marker ofovertly differentiated chondrocytes (Han Y and Lefebvre V, 2008Molecular and Cellular Biology, 28:4999-5013). These results indicatethat although day 3 cultures are entering into the chondrogenic lineage,they have not yet undergone overt differentiation into chondrocytes. Itshould also be noted that although day 3 cultures do not exhibitupregulated aggrecan expression, they do exhibit about a 4-foldupregulation in expression of transcripts for Col2a1 which encodescartilage-characteristic type II collagen (FIG. 7A). However, unlikeaggrecan, Col2a1 is expressed by prechondrogenic mesenchymal cells at anearly stage in the chondrogenic lineage in the developing limb, as wellas by differentiated chondrocytes (Han Y and Lefebvre V, 2008 Molecularand Cellular Biology, 28:4999-5013; Nah H D and Upholt W B, 1991 J BiolChem, 266:23446-23452; Sakai K et al., 2001 Matrix Biol, 19:761-767).The gene expression patterns indicate that day 3 cultures are enteringinto the chondrogenic lineage, but have not yet undergone overtdifferentiation into chondrocytes.

As shown in FIGS. 7A and 7B, day 4 cultures exhibit further upregulationin the expression of the transcriptional regulators Sox9 and Brachyury.Sox9 expression is more than 2-fold higher and Brachyury expression is5-fold higher in day 4 cultures than in day 3 cultures (FIGS. 7A and7B). Moreover, day 4 cultures exhibit a greater than 4-fold upregulationin the expression of the definitive cartilage marker aggrecan (FIG. 7A).The upregulated expression of aggrecan indicates that the day 4 culturesare in an early phase of overt differentiation into chondrocytes.

By day 7 of culture, the expression of Sox9 and Brachyury are strikinglydown-regulated (FIGS. 7A and 7B). On day 7, the expression of Sox9 isdecreased about 10-fold (FIG. 7A) and Brachyury expression about 24-fold(FIG. 7B) compared to day 4. By day 14, Sox9 expression continues todecline to very low levels and the expression of Brachyury on day 14 isnegligible (FIGS. 7A and 7B). The downregulation of Sox9 and Brachyuryexpression on day 7 and 14 is accompanied by an about 8-foldupregulation in the expression of the cartilage markers aggrecan andCol2a1. As described above, the upregulation of aggrecan and Col2a1expression at days 7 and 14 correlates with the uniform accumulation ofAlcian blue positive matrix and type II collagen detectable byimmunostaining. These gene expression and staining patterns indicatethat day 7 and 14 cultures have quite uniformly undergone overtdifferentiation into chondrocytes.

Significantly, the day 7 and 14 cultures which have undergone overtcartilage differentiation do not express markers of hypertrophicchondrocyte maturation. No expression of Col10a1, a definitive molecularmarker of hypertrophic chondrocytes, is detectable in day 7 or day 14cultures, and expression of osteopontin (OPN), a marker of terminalhypertrophic maturation is negligible (FIG. 7A). Day 7 and 14 culturesalso exhibit negligible expression of Indian hedgehog (Ihh), a marker ofprehypertrophic chondrocytes that have initiated maturation. Theseresults indicate that the chondrocytes comprising day 7 and 14 cultureshave not undergone hypertrophic chondrocyte maturation.

Inappropriate hypertrophic maturation is a hallmark of osteoarthritis;thus, the presence of this phenotype in the cell populations to be usedfor human (or other mammalian) therapy is undesirable. In some systems,chondrogenic cultures induced by BMPs show high expression of genesassociated with chondrocyte hypertrophy including collagen type (COL) Xand Indian hedgehog (1HH). Such hypertrophy-associated changes are foundin pathological conditions such as osteoarthritis. The absence ornegligible expression of such hypertrophy-associated markers is anindication that the cells described herein possess advantages over otherpreparations and are ideally suited for therapeutic administration todiseased or damaged cartilaginous joint tissues.

As shown in FIGS. 7C-7E, the expression of cartilage lineage markers inhESC micromass cultures treated with BMP2 alone were compared toexpression in cultures treated with TGFβ1 alone or treated with acombination of TGFβ1 and BMP2. Unlike BMP2, TGFβ1 alone does notstimulate the expression of cartilage markers. Indeed, TGFβ1-treatedcultures and untreated control cultures exhibit comparable low levels ofexpression of aggrecan, Sox9, and Brachyury (FIGS. 7C-7E).

Example 4 Differentiation of Undifferentiated Pluripotent hESCs/DirectAnd Progressive Differentiation Of Undifferentiated Pluripotent hESCsInto Progenitor Cells In Various Phases Of The Chondrogenic Lineage

Fulfilling the potential of hESCs for treatment of degenerative diseasesof cartilage such as osteoarthritis requires developing methods fordirecting their differentiation into the chondrogenic lineage. Describedabove are culture systems and conditions that allow hESCs toprogressively and uniformly differentiate into the chondrogenic lineage.As described above, undifferentiated pluripotent hESCs subjected tomicromass culture in the presence of BMP2 (or BMP2 and TGFβ together)directly undergo progressive and quite uniform differentiation into thechondrogenic lineage without prior EB formation.

The direct and progressive chondrogenic differentiation that pluripotentundifferentiated hESCs undergo has enabled the characterization ofprogenitor cells in different phases of the chondrogenic lineage whichcan be tested and compared for their abilities to repair damaged ordiseased human cartilage using cell based tissue engineering therapies.By gene expression profiling of BMP2-treated hESC micromass culturesharvested at different periods of culture, cells that are just enteringinto the chondrogenic lineage (day 3 cultures i.e., chondroprogenitors),cells which are in an early phase of overt chondrocyte differentiation(day 4 cultures i.e., early chondrocytes), and cells which haveuniformly undergone overt differentiation into chondrocytes, but havenot undergone hypertrophic maturation (day 7 and 14 cultures i.e., fullydifferentiated chondrocytes) were identified.

In particular, day 3 hESC micromass cultures are characterized byupregulated expression of the transcriptional regulators Sox9 andBrachyury, but do not exhibit upregulated expression of aggrecan, adefinitive highly specific molecular marker of overtly differentiatedchondrocytes, and exhibit little or no Alcian blue positive matrix.Thus, day 3 cultures have entered into the chondrogenic lineage, buthave not yet undergone overt differentiation into chondrocytes. Thischaracteristic of progenitor cells at such an early stage in thechondrogenic lineage is useful in repairing cartilage defects. Cells ina very early phase of the chondrogenic lineage are more responsive tosignals that promote their participation in repair compared to cells atlate stages of the lineage.

Cell populations at certain stages of development are preferred forcertain clinical indications. For example, day 2-4 cells (chondrogenicprogenitors or early chondrocytes) integrate better into damaged tissueand cartilage and elicit better and longer lasting repair.

Chondroprogenitors (day 2-3 cells) are preferentially used for limb orfinger regeneration, mensical or ligament repair, and fracture repair.Early chondrocytes (day 4 cells) are used for repair of articularcartilage defects due to injury, for repair or prevention of articularcartilage damage due to chronic disease (OA or RA), for regeneration andreplacement of lost cartilage tissue due to trauma or congenital defect,for meniscal repair, and for repair of fibrocartilage of thetemporomandibular joint. More fully differentiated chondrocytes aresuitable for indications such as for articular cartilage repairfollowing acute or traumatic injury, or for repair or prevention ofarticular cartilage due to disease (OA or RA), as well as for fracturerepair via endochondral ossification. Each stage is useful for modelingof cartilage development and differentiation, and iPSC derived cells inparticular are useful for patient specific repair and regeneration, aswell as for modeling of human disease, design and testing of targetedtherapeutics, and for disease-specific repair of genetically compromisedcartilage tissue.

Day 4 BMP2-treated hESC micromass cultures exhibit further upregulationin the expression of Sox9 and Brachyury, and exhibit a greater than4-fold upregulation in the expression of the definitive cartilage markeraggrecan, indicating that the day 4 cultures are in an early phase ofovert differentiation into chondrocytes. Day 7 and 14 cultures arecharacterized by a downregulation in expression of Sox9 and Brachyuryaccompanied by an about 8-fold upregulation in the expression of thecartilage markers aggrecan and Col2a1. A uniform Alcian blue positivematrix is also present throughout the extent of the day 7 and 14cultures and type II collagen detectable by immunostaining is presentthroughout the extent of the cultures. Day 7 and 14 cultures do notexpress markers of hypertrophic chondrocyte maturation such as Col10a1,Ihh, or osteopontin. Thus, day 7 and 14 cultures have undergone overtdifferentiation into chondrocytes, but have not undergone hypertrophicmaturation. This is significant in considering utilization of the cellsfor articular cartilage repair, as the chondrocytes comprising articularcartilage do not normally undergo hypertrophic maturation, and indeedinappropriate hypertrophic maturation of articular chondrocytes ischaracteristic of osteoarthritis. The fact that the chondrocytes of day7 and/or 14 cultures have not undergone hypertrophic maturation makesthese cells attractive candidates for repair of cartilage defects.

Micromass Cultures of hESCs for Identification of Factors Involved inthe Determination Process that Channels Progenitor Cells into theChondrogenic Lineage

Although some of the regulatory genes such as Sox9 that control theonset of cartilage differentiation have been identified, little is knownabout the genes and other factors that are involved in the progressivedetermination or commitment of embryonic progenitor cells into thechondrogenic lineage. The progressive differentiation of hESCs inmicromass culture in the presence of BMP2 provides suitable system fordefining factors involved in these early determinative events. Analysisof the transcriptomes of cells in various stages of their lineageprogression from an undifferentiated pluripotent hESC into a chondrocytefacilitates the identification of genes, signaling molecules, andregulatory networks that control early chondrogenic lineage commitmentevents, and enable delineation of unknown genes involved in thedetermination process.

The gene expression profiling described above indicates a role for thetranscription factor Brachyury in channeling cells into the chondrogeniclineage. Previous studies have shown that Brachyury, which is a T-boxtranscription factor, is highly expressed during gastrulation, and playsan essential role in primary mesoderm formation (Herrmann B G, 1995Seminars in Developmental Biology, 6:385-394). Later in development,Brachyury is expressed in the lateral mesoderm at the onset of limb budformation and subsequently by the prechondrogenic mesenchymal cells thatwill give rise to cartilage in the developing limb (Liu C et al., 2003Development, 130:1327-1337). As described above, expression of Brachyuryis upregulated (24-fold) as hESC micromass cultures treated with BMP2are just entering into the chondrogenic lineage as assayed byupregulated expression of the chondrogenic transcription factor Sox9.The concomitant upregulation of Brachyury and Sox9 expression occursbefore upregulation of the expression of the definitive cartilage markeraggrecan. The expression of Brachyury and Sox9 then concomitantlyincrease even further as the BMP2-treated hESC micromass culturesinitiate upregulated expression of aggrecan. After the onset ofchondrogenesis, the expression of Brachyury and Sox9 concomitantlydecrease to negligible levels as overt cartilage differentiation occurs.This expression pattern during the progressive differentiation ofBMP2-treated undifferentiated pluripotent hESCs into chondrocytesindicates that Brachyury is involved in regulating the early commitmentof mesodermal progenitor cells into the chondrogenic lineage.

It has been demonstrated that Brachyury expression is upregulated at theonset of the chondrogenic differentiation of the mesenchymal cell lineC3H10T1/2 in response to BMP2 signaling (Hoffmann A et al., 2002 J CellSci, 115:769-781), and forced expression of Brachyury promotes theexpression of Sox9 and the chondrogenic differentiation of C3H10T1/2cells (Hoffmann A et al., 2002 J Cell Sci, 115:769-781). Moreover, adominant negative form of Brachyury impairs BMP2-stimulated chondrogenicdifferentiation of C3H10T1/2 cells (Hoffmann A et al., 2002 J Cell Sci,115:769-781). It has been suggested that Brachyury may endowprechondrogenic progenitor cells such as C3H101/2 cells with the abilityto undergo chondrogenic differentiation in response to BMP2 (Hoffmann Aet al., 2002 J Cell Sci, 115:769-781). The results described aboveindicate that Brachyury expression is upregulated as hESCs enter intothe chondrogenic lineage in response to BMP2 is consistent with previousstudies. These studies indicate that Brachyury is involved in thedetermination process that channels progenitor cells into thechondrogenic lineage.

Example 5 Differentiation of Human Embryonic Stem Cells into theChondrogenic Lineage

As shown in FIG. 4C, extracellular cartilage-characteristic type IIcollagen is present throughout BMP2-treated hESC micromass cultures asdetected by immunostaining of sagittal sections of the cultures with atype II collagen antibody. In addition, cells whose cytoplasm stainswith a monoclonal antibody against the globular domain of the cartilagemarker aggrecan are present throughout BMP-2-treated hESC micromasscultures (FIG. 9), indicating that virtually all of the cells that aresurrounded by a type II collagen extracellular matrix are expressing thecartilage marker aggrecan as assayed by immunostaining with a cellautonomous marker.

The accumulation of cartilage matrix in cultures is routinely monitoredby whole mount staining of the intact cultures with Alcian blue, pH 1.0,which stains the sulfated proteoglycans of the matrix. Quite uniformaccumulation of Alcian blue stainable matrix is detectable by wholemount staining in hESC micromass cultures treated with either BMP2 aloneor BMP2 plus TGF-131 (FIGS. 4-6). To histologically evaluate the extentof cartilage differentiation, sagittal sections of hESC micromasscultures were stained with Alcian blue and counterstained with thenuclear stain Nuclear Fast Red. Cells surrounded by an Alcian bluestainable extracellular matrix are present throughout virtually theentire extent of histological sections through hESC micromass culturestreated with either BMP2 alone (FIG. 10A) or with BMP2 plus TGF-β1(FIGS. 10B and 10C). In addition to the extensive cartilage tissue, theBMP2-treated hESC cultures contained a small number of tubularstructures (FIG. 10A; arrows). The number of tubules that are detectableis considerably reduced when the cultures are supplied with TGF-131 aswell as BMP2, and the tubules constitute only a very small percentage ofthe cultures. Thus, although not completely homogeneous, chondrogenicdifferentiation in hESC micromass cultures treated with both BMP2 andTGF-β1 occurs quite uniformly and is considerably more robust andextensive than in other cultures systems that have been previouslyreported. Indeed, BMP2 TGF-β-treated micromass cultures established fromembryoid body (EB) cells are quite heterogeneous consisting not only ofchondrogenic tissue, but also a large amount of adipose tissue, as welltubules (FIG. 11).

Example 6 iPSC-Derived Chondrocytes for Regenerative Medicine and HumanDisease Modeling

iPSCs are obtained from a patients own cells, thereby offering a meansfor patient specific cell mediated therapy and regenerative medicine(Amabile G and Meissner A 2009 Trends Mol Med 15:59-68). As shown inFIGS. 11, 12, and 13, iPSC undergo directed and progressivedifferentiation into the chondrogenic lineage by the methods describedherein.

FIG. 11 (A) shows a control micromass culture of iPSC maintained withoutBMP2 supplementation for 14 days, which exhibits little Alcianblue-positive matrix accumulation. FIG. 11 (B) shows a day 14 BMP2treated iPSC micromass culture, which shows intense and widespreadAlcian blue staining present throughout the extent of the culture.

To verify the progression of chondrogenic differentiation by theBMP2-supplemented iPSC-derived micromass cultures, quantitativereal-time RT-PCR of the mRNA expression of marker genes characteristicof the chondrogenic lineage including Brachyury, sox9, Col2a1 andaggrecan was used. Levels of these marker genes underwent similarupregulation during the culture period as do hESC micromass culturessupplemented with BMP2. In addition, expression of Osteopontin mRNA, amarker of hypertrophic chondrocytes, was negligible in BMP2-supplementediPSC micromass cultures. Thus, micromass cultures of iPSC using thepresent methods undergo progressive differentiation into thechondrogenic lineage and also do not undergo hypertrophy in vitro.

Studies were carried out to compare chondrogenic differentiation bymicromass cultures established from iPSC dissociated to single cells bythree different enzymatic approaches: trypsin-EDTA, Accutase or TrypLESelect. As shown in FIG. 12, after 7 days, BMP2-supplemented micromasscultures established from iPSC previously dissociated with eithertrypsin-EDTA (FIG. 12A), TrypLE Select (FIG. 12B) or Accutase (FIG. 12C)each exhibit comparable accumulation of intensely stained and widespreadAlcian blue-positive matrix, indicating that each dissociation method isequally effective in producing single iPSC that can successfully undergochondrogenic differentiation in the protocol in response to BMP2. LittleAlcian blue positive matrix is present in cultures maintained withoutBMP2 supplementation (FIGS. 12D-F).

As shown in FIG. 13, iPSC micromass cultures undergo comparablechondrogenic differentiation as assayed by widespread and intense AlcianBlue staining after 14 days of culture in response to BMP2 (FIG. 13A) aswell as a combination of BMP2 and TGFβ1 (FIG. 13B), in contrast to iPSCmicromass cultures which received no BMP2 supplementation whichaccumulate little Alcian blue-positive matrix (FIG. 13C).

Thus, the method is useful for generation of iPSC for patient specificcartilage repair and restoration therapies including articular cartilagedefect repair of joints due to injury or chronic disease such as OA orRA; and for limb regeneration, meniscal or ligament defect repair, andfracture repair, using approaches described above.

iPSC obtained from individuals with genetic disorders provide a meansfor disease modeling and screening for the design and testing oftargeted therapeutics (Laustriat D et al., 2010 Biochem Soc Trans38:1051-1057; Lengner C J 2010 Ann NY Acad Sci 1192: 38-44). iPSC whencombined with gene targeting provide an approach for monogenic diseasetreatment (Wong G K Y and Chiu A T 2010 Biotechnol Adv 28:715-724).

As shown in FIG. 16, iPSC were generated from a human patient withchondrodysplasia. iPSC from human patients with genetic cartilagedisorders are induced to differentiate into chondrogenic lineage usingthe methods described herein. The high density culture methods,including the high density micromass approach which was used to inducechondrogenic differentiation of hESC and iPSC is suitable for use as atoxicology test (Piersma AH 2004 Toxicol Lett 149:147-153; Ponce R A2001 Curr Protoc Toxicol 13: 13.3); as a genomic screen to identifynovel genetic regulators of cartilage development (James C G et at.,2005 Mol Biol Cell 16:5316-5333), for detection of developmental toxinsaffecting chondrogenesis (Hanse et al 2001 Free Radic Biol Med 31:1582-1592) and for mechanistic evaluation of potential skeletaldysplasia therapeutics (Woods A et al., 2007 Endocrinol 148: 5030-5041).iPSC-derived from patients with genetic disorders of cartilage (e.g.,chondrodysplasia or achondroplasia) and induced to undergo chondrogenicdifferentiation by the methods described herein are useful for modelingof the disease process and design and testing of drug therapies. Geneprofiling of the iPSC-derived chondrodysplastic chondrogenic cells isused to identify key genes and factors which mediate the diseaseprocess, which provides disease-specific targets for therapeuticintervention. Targeted therapeutics for the disease are tested in vitroby determining the response of the diseased iPSC-derived chondrogeniccells produced by the method to the agents in vitro. iPSC-deriveddisease-specific chondrogenic cells produced by the method are also usedin conjunction with gene therapy as an approach to restore normalcartilage structure or function in the genetically-diseased patients.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method for direct differentiation of human embryonic stem cells(hESCs) into chondrogenic cells comprising: providing a population ofhESCs on a substrate, said population being substantially free ofembryoid bodies (EBs); detaching said hESCs from said substrate anddissociating said hESCs; establishing a high-density micromass cultureof said hESCs; contacting said population with a bone morphogeneticprotein; wherein said hESCs directly differentiate into a substantiallyuniform population of chondrogenic cells.
 2. The method of claim 1,wherein said substrate is a feeder layer comprising mouse embryonicfibroblasts (MEF).
 3. The method of claim 1, wherein said substrate isMatrigel or other polymeric or gelatinous substrates or scaffolds. 4.The method of claim 1, further comprising administering transforminggrowth factor beta to said hESCs.
 5. The method of claim 1, wherein saidBMP comprises BMP-2 and wherein said BMP-2 is administered about 24hours after establishing said high-density micromass culture.
 6. Themethod of claim 1, wherein said BMP comprises BMP-2 and wherein saidBMP-2 is administered about 48 hours after establishing saidhigh-density micromass culture.
 7. The method of claim 1, wherein saidhESCs differentiate into fully differentiated chondrocytes within about14 days.
 8. The method of claim 1, wherein said hESCs differentiate intofully differentiated chondrocytes within about 7 days.
 9. The method ofclaim 1, wherein said hESCs differentiate into chondroprogenitor cellswithin about 2-3 days.
 10. The method of claim 1, wherein said hESCsdifferentiate into early chondrocytes within about 4 days.
 11. Themethod of claim 1, wherein at least about 85% of said hESCsdifferentiate into cells of the chondrogenic lineage.
 12. The method ofclaim 1, wherein at least about 95% of said hESCs differentiate intocells of the chondrogenic lineage.
 13. The method of claim 1, whereinsaid hESCs are dissociated with trypsin, TrypLE Select, or Accutase. 14.The method of claim 1, further comprising the administration ofRho-associated kinase (ROCK) inhibitor.
 15. A method for directdifferentiation of induced pluripotent stem cells (iPSCs) intochondrogenic cells comprising: providing a population of iPSCs on asubstrate, said population being substantially free of EBs; detachingsaid iPSCs from said substrate and dissociating said iPSCs; establishinga high-density culture of said iPSCs; contacting said iPSCs with a bonemorphogenetic protein thereby directing the differentiation of iPSCsinto the chondrogenic lineage.
 16. The method of claim 1 or 15, whereinsaid hESCs are in the form of a micromass suspension culture or a pelletculture.
 17. A population of chondrogenic cells, wherein said populationis substantially free of non-chondrogenic cells, said population havingbeen differentiated from ESCs by the method of claim
 1. 18. A populationof chondrogenic cells, wherein said population is substantially free ofnon-chondrogenic cells, said population having been differentiated fromiPSCs by the method of claim
 15. 19. A uniformly differentiatedpopulation of chondrogenic cells, at least 95% of said cells being at asingle defined stage of differentiation, wherein said population issubstantially free of fully differentiated chondrocytes.
 20. Thepopulation of claim 19, wherein said stage is selected from the groupconsisting of day 2 or 3 chondrogenic cells (chondroprogenitors), or day4 chondrogenic cells (early chondrocytes).
 21. A method of repairing orrestoring cartilage, comprising contacting damaged or diseased cartilagewith the population of claim
 17. 22. A method of preventing or treatingarthritis, comprising administering to an articulating joint thepopulation of claims
 17. 23. A method of treating fracture repaircomprising contacting a bone fracture site with the population of claim17.
 24. A method of treating or repairing torn or ruptured ligaments ormenisci comprising administering to articulating joints the populationof claim
 17. 25. A method of regenerating fingers or limbs with thepopulation of claim
 17. 26. A method of identifying a gene involved inthe development of a cartilage disorder, comprising providing thepopulation of claim 18 and detecting an increase or decrease in geneexpression compared to normal control cells, wherein said increase ordecrease indicates that said gene is involved in the development of saidcartilage disorder.
 27. The method of claim 26, wherein said cartilagedisorder comprises chondrodysplasia or achondroplasia.
 28. A method ofidentifying a cartilage promoting agent, comprising contacting thepopulation of claim 18 with a candidate compound and detectingchondrogenesis, wherein an increase in chondrogenesis in the presence ofthe said compound compared to in the absence of said compound indicatesthat said compound promotes the formation of cartilage.
 29. The methodof claim 15, wherein said substrate is a feeder layer comprising mouseembryonic fibroblasts (MEF).
 30. The method of claim 15, wherein saidsubstrate is Matrigel or other polymeric or gelatinous substrates orscaffolds.
 31. The method of claim 15, further comprising administeringtransforming growth factor beta to said hESCs.
 32. The method of claim15, wherein said BMP-2 is administered about 24 hours after establishingiPSC high-density micromass culture.
 33. The method of claim 15, whereinsaid BMP-2 is administered 48 hours after initiation of said iPSChigh-density micromass culture.
 34. The method of claim 15, wherein saidiPSCs differentiate into full differentiated chondrocytes within about14 days.
 35. The method of claim 15, wherein said iPSCs differentiateinto fully differentiated chondrocytes within 7 days.
 36. The method ofclaim 15, wherein said iPSCs differentiate into chondroprogenitor cellswithin about 2-3 days.
 37. The method of claim 15, wherein said iPSCsdifferentiate into early chondrocytes within about 4 days.
 38. Themethod of claim 15, wherein at least about 85% of said iPSCsdifferentiate into cells of the chondrogenic lineage.
 39. The method ofclaim 15, wherein at least about 95% of said iPSCs differentiate intocells of the chondrogenic lineage.
 40. The method of claim 15, whereinsaid iPSCs are dissociated with trypsin, TrypLE Select, or Accutase. 41.The method of claim 15, further comprising the administration ofRho-associated kinase (ROCK) inhibitor.